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Current malaria diagnostics are invasive, lack sensitivity, and rapid tests are plagued by deletions in target antigens. Here we introduce the Cytophone, an innovative photoacoustic flow cytometer platform with high-pulse-rate lasers and a focused ultrasound transducer array to noninvasively detect and identify malaria-infected red blood cells (iRBCs) using specific wave shapes, widths, and time delays generated from the absorbance of laser energy by hemozoin, a universal biomarker of malaria infection. In a population of Cameroonian adults with uncomplicated malaria, we assess our device for safety in a cross-sectional cohort (n = 10) and conduct a performance assessment in a longitudinal cohort (n = 20) followed for 30 ± 7 days after clearance of parasitemia. Longitudinal cytophone measurements are compared to point-of-care and molecular assays (n = 94). Cytophone is safe with 90% sensitivity, 69% specificity, and a receiver-operator-curve-area-under-the-curve (ROC-AUC) of 0.84, as compared to microscopy. ROC-AUCs of Cytophone, microscopy, and RDT compared to quantitative PCR are not statistically different from one another. The ability to noninvasively detect iRBCs in the bloodstream is a major advancement which offers the potential to rapidly identify both the large asymptomatic reservoir of infection, as well as diagnose symptomatic cases without the need for a blood sample., (© 2024. The Author(s).)

Objectives: The isolated Príncipe is at the malaria pre-elimination stage. Autochthonous clinical cases have been reported sporadically on the island, signaling the possibility of a sizable subpatent (i.e., rapid diagnostic test- and microscopy-negative and polymerase chain reaction [PCR]-positive) parasite reservoir., Methods: Asymptomatic low-density infections were detected by quantitative PCR (qPCR) targeting Plasmodium falciparum multicopy genes (pfr364 and varATS). Positivity rates were assayed for samples surveyed by active case detection (n = 112) and reactive case detection (n = 221) in 2022., Results: qPCR unveiled 70% of low parasitemia carriers, reaching >90% in reactive case detection. The high P. falciparum prevalence was confirmed by the two high-sensitivity qPCR protocols. Higher positivity rates were observed in the localities where most malaria cases were reported in 2022. Most parasitemias were very low (<2 Pf /µl)., Conclusions: These findings suggest that pre-elimination surveillance can benefit from the routine application of highly sensitive tools to unveil otherwise invisible but potentially relevant parasite populations., Competing Interests: Declarations of competing interest The authors have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: According to the World Health Organization, Microscopy is the gold standard for diagnosing malaria. However, the performance of this examination depends on the experience of the microscopist and the level of parasitemia. Thus, molecular biology detection of malaria could be an alternative technique., Aim: evaluate the contribution of molecular biology in detecting imported malaria., Methods: This was a descriptive, prospective study, including all students, from the Monastir region, and foreigners, from countries endemic to malaria. The study period was from September 2020 to April 2021. Each subject was screened for malaria by three methods: direct microscopic detection of Plasmodium, detection of plasmodial antigens, and detection of plasmodial DNA by nested PCR., Results: Among the 127 subjects screened, only one had a positive microscopic examination for Plasmodium falciparum. Among the 126 subjects with a negative microscopic examination, twelve students had a positive nested PCR result, i.e. 9.5%. Molecular sequencing allowed the identification of ten isolates of Plasmodium falciparum, one Plasmodium malariae and one Plasmodium ovale. Our study showed that the results of nested PCR agreed with those of microscopy in 90.6% of cases., Conclusion: Nested PCR seems more sensitive for the detection of low parasitemias. Hence the importance of including molecular biology as a malaria screening tool to ensure better detection of imported cases.

Background: Application of numerous malaria control interventions has led to reduction in clinical malaria cases and deaths but also the realisation that asymptomatic parasite carriers play a key role in sustaining transmission. This study assessed the effectiveness of using the Ultra-sensitive NxTek eliminate RDT (uRDT) and conventional SD Bioline HRP2 RDT (cRDT) in diagnosing asymptomatic parasitaemia while measuring the impact of mass testing, treatment and tracking (MTTT) on the prevalence of asymptomatic malaria over a 1-year period in Ghana., Methods: A total of 4000 targeted participants from two towns, Obom and Kofi Kwei, with their surrounding villages, were tested for asymptomatic malaria four times over the study period using uRDT (intervention) and the cRDT (control) respectively. Participants carrying malaria parasites were followed by home visit and phone calls for compliance to treatment, and filter paper blood blots collected from participants were used to determine true parasite carriage by PET-PCR. A mathematical model of the study site was developed and used to test the impact of test sensitivity and mass migration on the effect of MTTT., Results: The start and end point sensitivities of the cRDT were 48.8% and 41.7% and those for the uRDT were 52.9% and 59.9% respectively. After a year of MTTTs, asymptomatic parasite prevalence, as determined by PCR, did not differ statistically in the control site (40.6% to 40.1%, P = 0.730) but decreased at the intervention site (55.9% to 46.4%, P < 0.0001). Parasite prevalence by RDT, however, indicated statistical reduction in the control site (25.3% to 22.3%, P = 0.017) and no change in the intervention site (35.1% to 36.0%, P = 0.614). The model predicted a mild effect of both diagnostic sensitivity and human movement in diminishing the impact of MTTT in the study sites., Conclusions: Asymptomatic parasite prevalence at the molecular level reduced significantly in the site where the uRDT was used but not where the cRDT was used. Overall, the uRDT exhibited higher sensitivity relative to the cRDT. Highly sensitive molecular techniques such as PET-PCR should be included in parasite prevalence estimation during MTTT exercises., (© 2024. The Author(s).)

Background: Effective testing for malaria, including the detection of infections at very low densities, is vital for the successful elimination of the disease. Unfortunately, existing methods are either inexpensive but poorly sensitive or sensitive but costly. Recent studies have shown that mid-infrared spectroscopy coupled with machine learning (MIRs-ML) has potential for rapidly detecting malaria infections but requires further evaluation on diverse samples representative of natural infections in endemic areas. The aim of this study was, therefore, to demonstrate a simple AI-powered, reagent-free, and user-friendly approach that uses mid-infrared spectra from dried blood spots to accurately detect malaria infections across varying parasite densities and anaemic conditions., Methods: Plasmodium falciparum strains NF54 and FCR3 were cultured and mixed with blood from 70 malaria-free individuals to create various malaria parasitaemia and anaemic conditions. Blood dilutions produced three haematocrit ratios (50%, 25%, 12.5%) and five parasitaemia levels (6%, 0.1%, 0.002%, 0.00003%, 0%). Dried blood spots were prepared on Whatman ™ filter papers and scanned using attenuated total reflection-Fourier Transform Infrared (ATR-FTIR) for machine-learning analysis. Three classifiers were trained on an 80%/20% split of 4655 spectra: (I) high contrast (6% parasitaemia vs. negative), (II) low contrast (0.00003% vs. negative) and (III) all concentrations (all positive levels vs. negative). The classifiers were validated with unseen datasets to detect malaria at various parasitaemia levels and anaemic conditions. Additionally, these classifiers were tested on samples from a population survey in malaria-endemic villages of southeastern Tanzania., Results: The AI classifiers attained over 90% accuracy in detecting malaria infections as low as one parasite per microlitre of blood, a sensitivity unattainable by conventional RDTs and microscopy. These laboratory-developed classifiers seamlessly transitioned to field applicability, achieving over 80% accuracy in predicting natural P. falciparum infections in blood samples collected during the field survey. Crucially, the performance remained unaffected by various levels of anaemia, a common complication in malaria patients., Conclusion: These findings suggest that the AI-driven mid-infrared spectroscopy approach holds promise as a simplified, sensitive and cost-effective method for malaria screening, consistently performing well despite variations in parasite densities and anaemic conditions. The technique simply involves scanning dried blood spots with a desktop mid-infrared scanner and analysing the spectra using pre-trained AI classifiers, making it readily adaptable to field conditions in low-resource settings. In this study, the approach was successfully adapted to field use, effectively predicting natural malaria infections in blood samples from a population-level survey in Tanzania. With additional field trials and validation, this technique could significantly enhance malaria surveillance and contribute to accelerating malaria elimination efforts., (© 2024. The Author(s).)

Herein, we report a case of uncomplicated falciparum malaria with late parasitological failure in a 45-year-old businessman returning from Ghana. The patient visited the emergency department with high fever, headache, and dizziness. He traveled without antimalarial chemoprophylaxis. Laboratory tests led to the diagnosis of uncomplicated falciparum malaria with an initial density of 37,669 parasites per μL of blood (p/μL). The patient was treated with intravenous artesunate followed by atovaquone/proguanil. He was discharged with improved condition and decreased parasite density of 887 p/μL. However, at follow-up, parasite density increased to 7,630 p/μL despite the absence of any symptoms. Suspecting treatment failure, the patient was administered intravenous artesunate and doxycycline for seven days and then artemether/lumefantrine for three days. Blood smear was negative for asexual parasitemia after re-treatment but positive for gametocytemia until day 101 from the initial diagnosis. Overall, this case highlights the risk of late parasitological failure in patients with imported uncomplicated falciparum malaria., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2024 The Korean Academy of Medical Sciences.)

Malaria is a deadly disease that is transmitted through mosquito bites. Microscopists use a microscope to examine thin blood smears at high magnification (1000x) to identify parasites in red blood cells (RBCs). Estimating parasitemia is essential in determining the severity of the Plasmodium falciparum infection and guiding treatment. However, this process is time-consuming, labor-intensive, and subject to variation, which can directly affect patient outcomes. In this retrospective study, we compared three methods for measuring parasitemia from a collection of anonymized thin blood smears of patients with Plasmodium falciparum obtained from the Clinical Department of Parasitology-Mycology, National Reference Center (NRC) for Malaria in Paris, France. We first analyzed the impact of the number of field images on parasitemia count using our framework, MALARIS, which features a top-classifier convolutional neural network (CNN). Additionally, we studied the variation between different microscopists using two manual techniques to demonstrate the need for a reliable and reproducible automated system. Finally, we included thin blood smear images from an additional 102 patients to compare the performance and correlation of our system with manual microscopy and flow cytometry. Our results showed strong correlations between the three methods, with a coefficient of determination between 0.87 and 0.92., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Acherar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Microscopy continues to be the mainstay for the evaluation of parasitaemia in malaria but requires laboratory support and microbiological experience. Other fast and simple methods are necessary., Methods: A retrospective observational study of imported malaria treated from July-2007 to December-2020 was carried out to evaluate the association between the degree of parasitaemia and both rapid diagnostic tests (RDT) reactivity patterns and haematological parameters. Plasmodium falciparum monoinfections diagnosed by peripheral blood smear and/or polymerase chain reaction (PCR),which also had a positive RDT result in the same blood sample, were included in the study., Results: A total of 273 patients were included. Most of them were male (n = 256; 93.8%) and visiting friends and relatives (VFR) travellers (n = 252; 92.3%). Patients with plasmodial lactate dehydrogenase (pLDH) or aldolase and histidine-rich protein 2 (HRP-2) co-reactivity (Pan/Pf pattern) had a parasitaemia range between 0 and 37% while those with just HRP-2 reactivity (P. falciparum pattern) had ranges between 0 and 1%. Not a single case of P. falciparum pattern was found for parasitaemia ranges greater than 1%, showing a negative predictive value of 100% for high parasitaemia. All the correlations between haematological parameters and parasitaemia resulted to be weak, with a maximum rho coefficient of -0.35 for lymphocytes and platelets, and of 0.40 for neutrophils-to-lymphocytes count ratio. Multivariate predictive models were constructed reflecting a poor predictive capacity., Conclusions: The reactivity pattern of RDT allows a rapid semi-quantitative assessment of P. falciparum parasitaemia in travellers with imported malaria, discriminating patients with lower parasite loads. Haematological parameters were not able to estimate parasitaemia with sufficient precision., (© 2023. The Author(s).)

The "Lysis and Concentration Technique" (LCTe) involves lysing red blood cells and concentrating parasites to increase the chances of visualization in low parasitemia and in scenarios of evaluators with less knowledge. The lysis of red blood cells reduced the time of diagnosis by 21 s, showing that the effect produced by the treatment is comparable to the effect of the experience in parasitological examination. In addition, the concentration of parasites was 39.18% higher for slides with high parasitemia and 131.03% for slides with low parasitemia in relation to the standard slide. LCTe proved to be inexpensive, with a total cost of approximately US$0.07 per slide made, which allows it to be easily implemented in most laboratories., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objectives: Optical spectrophotometry has been explored to quantify Plasmodium falciparum malaria parasites at low parasitemia, with potential to overcome the limitations of detection in the current diagnostic methods. This work presents the design, simulation and fabrication of a CMOS microelectronic detection system to automatically quantify the presence of malaria parasites in a blood sample., Methods: The designed system is composed by an array of 16 n+/p-substrate silicon junction photodiodes as photodetectors and 16 current to frequency (IF) converters. An optical setup was used to individually and jointly characterize the entire system., Results: The IF converter was simulated and characterized in Cadence Tools using UMC 1180 MM/RF technology rules, featuring a resolution of 0.01 nA, a linearity up to 1800 nA and a sensitivity of 4430 Hz/nA. After fabrication in a silicon foundry, the photodiodes' characterization presented a responsivity peak of 120 mA/W (λ = 570 nm) and a dark current of 7.15 pA at 0 V. Regarding the IF converter, it exhibited high linearity (R 2 ≈ 0.999) up to 30 nA, with a sensitivity of 4840 Hz/nA. Furthermore, the microsystem performance was validated using RBCs (Red Blood Cells) infected with P. falciparum and diluted at different parasitemia (12, 25 and 50 parasites/μL)., Conclusion: The microsystem was able to distinguish between healthy and infected RBCs, with a sensitivity of 4.5 Hz/parasites.μL -1 ., Significance: The developed microsystem presents a competitive result, when compared to the gold standard diagnosis methods, with increased potential for malaria in field diagnosis.

Background: High-quality malaria diagnosis is essential for effective treatment and clinical disease management. Microscopy and rapid diagnostic tests are the conventional methods performed as first-line malaria diagnostics in non-endemic countries. However, these methods lack the characteristic to detect very low parasitaemia, and accurate identification of the Plasmodium species can be difficult. This study evaluated the performance of the MC004 melting curve-based qPCR for the diagnosis of malaria in routine clinical practice in non-endemic setting., Methods and Results: Whole blood samples were collected from 304 patients with clinical suspicion of malaria and analysed by both the MC004 assay and conventional diagnostics. Two discrepancies were found between the MC004 assay and microscopy. Repeated microscopic analysis confirmed the qPCR results. Comparison of the parasitaemia of nineteen Plasmodium falciparum samples determined by both microscopy and qPCR showed the potential of the MC004 assay to estimate the parasite load of P. falciparum. Eight Plasmodium infected patients were followed after anti-malarial treatment by the MC004 assay and microscopy. The MC004 assay still detected Plasmodium DNA although no parasites were seen with microscopy in post-treatment samples. The rapid decline in Plasmodium DNA showed the potential for therapy-monitoring., Conclusion: Implementation of the MC004 assay in non-endemic clinical setting improved the diagnosis of malaria. The MC004 assay demonstrated superior Plasmodium species identification, the ability to indicate the Plasmodium parasite load, and can potentially detect submicroscopic Plasmodium infections., (© 2023. The Author(s).)

Hyperparasitaemia is an important event in the cascade of Plasmodium falciparum severe malaria (SM), and may also lead to SM associated complications and death, if left untreated. Here, we report two hyperparasitaemic patients with no life-threatening complications. Malaria diagnosis was performed using thick and thin blood smears and immunochromatographic-based rapid diagnostic tests (RDTs) purchased from three different manufacturers. Parasitaemia was calculated following the World Health Organization (WHO) guidelines. Haematological and biochemical investigations were also performed. Weekly follow-up of blood smear examination, blood pressure and temperature were recorded up to day 63. The first patient had 42% parasitaemia (100% asexual parasites). The second patient had 9.5% parasitaemia, comprising 46% asexual and 54% sexual stages, with a 1:1 male to female ratio. On the day of admission, both had presented abnormal haematological and biochemical parameters compared to the reference values. Remarkably, both the patients recovered successfully with oral artemisinin-based combination therapy (ACT) and a single dose of primaquine on day 1. Weekly follow-up did not show any parasite suggesting successful treatment with ACT without any side effects. The presence of hypergametocytaemia may hinder malaria elimination efforts, if not treated immediately., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Benudhar Mukhi, Himanshu Gupta, Kishore Punnath, Anupkumar R Anvikar, Bina Srivastava, Susanta Kumar Ghosh.)

Malaria elimination is a major goal to be reached in the next decade. Significant progress were made in the past, and the prevalence decreased in many areas while the positive trend stalled in the last years. The exact number of asymptomatic carriers of Plasmodium parasites is unknown since this population is not detected by conventional diagnosis methods and participate in the maintenance of transmission. Molecular methods to detect low parasitemia are not available at point-of-care in low-income countries of malaria endemic areas. Adaptation of molecular methods such as loop-mediated isothermal amplification of DNA may provide effective tools but it required simplification of DNA detection. Square waves voltammetry, easily imbedded in small device such as cell phone, was largely described for DNA detection but support for reaction was an issue to address. Here we used an efficient functionalization method of paper-based material to facilitate the interactions between isothermal amplification product and methylene blue for easy-to-use DNA detection. The proof-of-concept of qualitative detection of very low parasitemia from malaria infected patients using newly chemically treated paper for square waves voltammetry was obtained with a sensitivity and specificity of 100% and a limit-of-detection of 0.1 parasite. μL -1 corresponding to a parasitemia of 0.000002%., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Rapid and accurate tools are needed for high-throughput in vitro antibabesial drug testing. In this study, flow cytometry for the measuring of Babesia bovis in vitro culture, was developed using SYBR Green I and compared against the results of fluorescence-based assay and microscopic assay. A high correlation of measured parasitemia was observed with high R 2 value (R 2  = 0.9991) between flow cytometry and microscopic analysis. The degree of antibabesial drug sensitivity against B. bovis determined by flow cytometry was 0.424 ± 0.173 μM. Similar to the results of previously published studies involving fluorescence spectrometry-based assay (0.408 ± 0.011 μM) and microscopy-based assay (0.400 ± 0.017 μM). The outcomes of this present study suggest that flow cytometry assay using SYBR Green I can potentially be useful in determining parasitemia and can serve as a rapid alternative method to antibabesial drug testing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Malaria kills a child in sub-Saharan Africa every 2 min despite widely available interventions including intermittent preventive treatment in infants (IPTi). Since 2010, when World Health Organization (WHO) recommended IPTi, no country has implemented it. To our knowledge, no IPTi study has been conducted in Nigeria. Considering severity of malaria in infancy and urgency to improve malaria prevention, we proposed a study to investigate the efficacy of this intervention in reducing malarial morbidity and mortality., Objective(s): The aim of this was to determine the safety and efficacy of SP-IPTi in reducing the prevalence of asymptomatic malaria parasitemia and malarial-associated hospital admissions., Methods: We performed a cluster-randomized controlled trial in 1379 infants. SP was administered alongside routine vaccinations in immunization centers randomized to intervention groups. Infants in control groups received only routine vaccines. Malarial 'morbidity and adverse events were monitored through passive case-detection and cross-sectional surveys'., Results: SP-IPTi was safe. There was no statistically significant difference in terms of risks of asymptomatic parasitemia at 9 months, fever or hospitalization between our control and intervention groups., Conclusions: Our study demonstrated that SP-IPTi had no benefit but was well tolerated. WHO and some researchers have also reported declining efficacy of SP, due to increasing drug resistance., (© The Author(s) [2023]. Published by Oxford University Press.)

Individuals infected with HIV-1 experience more frequent and more severe episodes of malaria and are likely to harbor asymptomatic parasitemia, thus potentially making them more efficient reservoirs of malaria. Two studies (cross-sectional and longitudinal) were designed in sequence between 2015-2018 and 2018-2020, respectively, to test the hypothesis that HIV-1 infected individuals have higher prevalence of asymptomatic parasitemia and gametocytemia than the HIV-1 negatives. This article describes the overall design of the two studies, encompassing data for the longitudinal study and additional data to the previously published baseline data for the cross-sectional study. In the cross-sectional study, HIV-1 positive participants were significantly older, more likely to be male, and more likely to have parasitemia relative to HIV-1 negatives (P < 0.01). In the longitudinal study, 300 participants were followed for 6 months. Of these, 102 were HIV-1 negative, 106 were newly diagnosed HIV-1 positive, and 92 were HIV-1 positive and on antiretroviral therapy, including antifolates, at enrollment. Overall parasitemia positivity at enrollment was 17.3% (52/300). Of these, 44% (23/52) were HIV-1 negative, 52% (27/52) were newly diagnosed HIV-1 positives, and only 4% (2/52) were HIV-1 positive and on treatment. Parasitemia for those on stable antiretroviral therapy was significantly lower (hazard ratio: 0.51, P < 0.001), compared with the HIV-1-negatives. On follow-up, there was a significant decline in parasitemia prevalence (hazard ratio: 0.74, P < 0.001) among the HIV patients newly initiated on antiretroviral therapy including trimethoprim-sulfamethoxasole. These data highlight the impact of HIV-1 and HIV treatment on asymptomatic parasitemia over time.

Background: Detection of malaria parasitaemia in samples that are negative by rapid diagnostic tests (RDTs) requires resource-intensive molecular tools. While pooled testing using a two-step strategy provides a cost-saving alternative to the gold standard of individual sample testing, statistical adjustments are needed to improve accuracy of prevalence estimates for a single step pooled testing strategy., Methods: A random sample of 4670 malaria RDT negative dried blood spot samples were selected from a mass testing and treatment trial in Asembo, Gem, and Karemo, western Kenya. Samples were tested for malaria individually and in pools of five, 934 pools, by one-step quantitative polymerase chain reaction (qPCR). Maximum likelihood approaches were used to estimate subpatent parasitaemia (RDT-negative, qPCR-positive) prevalence by pooling, assuming poolwise sensitivity and specificity was either 100% (strategy A) or imperfect (strategy B). To improve and illustrate the practicality of this estimation approach, a validation study was constructed from pools allocated at random into main (734 pools) and validation (200 pools) subsets. Prevalence was estimated using strategies A and B and an inverse-variance weighted estimator and estimates were weighted to account for differential sampling rates by area., Results: The prevalence of subpatent parasitaemia was 14.5% (95% CI 13.6-15.3%) by individual qPCR, 9.5% (95% CI (8.5-10.5%) by strategy A, and 13.9% (95% CI 12.6-15.2%) by strategy B. In the validation study, the prevalence by individual qPCR was 13.5% (95% CI 12.4-14.7%) in the main subset, 8.9% (95% CI 7.9-9.9%) by strategy A, 11.4% (95% CI 9.9-12.9%) by strategy B, and 12.8% (95% CI 11.2-14.3%) using inverse-variance weighted estimator from poolwise validation. Pooling, including a 20% validation subset, reduced costs by 52% compared to individual testing., Conclusions: Compared to individual testing, a one-step pooled testing strategy with an internal validation subset can provide accurate prevalence estimates of PCR-positivity among RDT-negatives at a lower cost., (© 2022. The Author(s).)

Trypanosoma cruzi infection has expanded globally through human migration. In Spain, the mother-to-child route is the mode of transmission contributing to autochthonous Chagas disease (CD); however, most people acquired the infection in their country of origin and were diagnosed in the chronic phase (imported chronic CD). In this context, we assessed the quantitative potential of the Loopamp Trypanosoma cruzi detection kit (Sat-TcLAMP) based on satellite DNA (Sat-DNA) to determine parasitemia levels compared to those detected by real-time quantitative PCRs (qPCRs) targeting Sat-DNA (Sat-qPCR) and kinetoplast DNA minicircles (kDNA-qPCR). This study included 173 specimens from 39 autochthonous congenital and 116 imported chronic CD cases diagnosed in Spain. kDNA-qPCR showed higher sensitivity than Sat-qPCR and Sat-TcLAMP. According to all quantitative approaches, parasitemia levels were significantly higher in congenital infection than in chronic CD (1 × 10 -1 to 5 × 10 5 versus >1 × 10 -1 to 6 × 10 3 parasite equivalents/mL, respectively [ P  < 0.001]). Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR results were equivalent at high levels of parasitemia ( P  = 0.381). Discrepancies were significant for low levels of parasitemia and older individuals. Differences between Sat-TcLAMP and Sat-qPCR were not qualitatively significant, but estimations of parasitemia using Sat-TcLAMP were closer to those by kDNA-qPCR. Parasitemia changes were assessed in 6 individual cases in follow-up, in which trends showed similar patterns by all quantitative approaches. At high levels of parasitemia, Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR worked similarly, but significant differences were found for the low levels characteristic of late chronic CD. A suitable harmonization strategy needs to be developed for low-level parasitemia detection using Sat-DNA- and kDNA-based tests. IMPORTANCE Currently, molecular equipment has been introduced into many health care centers, even in low-income countries. PCR, qPCR, and loop-mediated isothermal amplification (LAMP) are becoming more accessible for the diagnosis of neglected infectious diseases. Chagas disease (CD) is spreading worldwide, and in countries where the disease is not endemic, such as Spain, the parasite Trypanosoma cruzi is transmitted from mother to child (congenital CD). Here, we explore why LAMP, aimed at detecting T. cruzi parasite DNA, is a reliable option for the diagnosis of congenital CD and the early detection of reactivation in chronic infection. When the parasite load is high, LAMP is equivalent to any qPCR. In addition, the estimations of T. cruzi parasitemia in patients living in Spain, a country where the disease is not endemic, resemble natural evolution in areas of endemicity. If molecular tests are introduced into the diagnostic algorithm for congenital infection, early diagnosis and timely treatment would be accomplished, so the interruption of vertical transmission can be an achievable goal.

Background: The recent worldwide increase in malaria cases highlights the need for renewed efforts to eliminate malaria. The World Health Organization advocates that malaria surveillance becomes a core intervention. Current methods to estimate the malaria burden rely on clinical malaria case reports and surveys of asymptomatic parasite infection mainly from children  < 5 years. In this study the hypothesis was that screening blood donors for malaria parasites would provide real-time information on the asymptomatic reservoir of parasites in the adult population and mirror other surveillance data., Methods: This study was conducted in Malawi, a high malaria burden country, at the Malawi Blood Transfusion Service, which collects blood units at donation sites countrywide. A secondary analysis was conducted on data obtained from a prior Sysmex XN-31 analyser malaria diagnostic evaluation study utilizing residual donor blood samples. XN-31 malaria results, donor age, sex, geographical location, and collection date, were analysed using standard statistical methods., Results: The malaria parasite prevalence in blood donors was 11.6% (614/5281 samples) increasing seasonally from December (8.6%) to April (18.3%). The median age was 21 years and 45.9% of donors were from urban areas, which showed a lower prevalence compared to non-urban regions. The Central administrative region had the highest and the Northern region the lowest malaria parasite prevalence. The donors were predominantly male (80.2%), 13.1% of whom had malaria parasites, which was significantly higher (p < 0.0001) than for female donors (7.4%). Multivariable logistic regression analysis showed that age, location, and collection month were significant predictors of malaria positivity in males, whereas in females only location was significant. There was no gender difference in parasite density nor gametocyte carriage., Conclusions: This study demonstrates the powerful utility of screening blood donors for malaria parasites using the XN-31, which not only improves the safety of blood transfusion, but provides valuable complementary surveillance data for malaria control, especially targeting males, who are generally excluded from periodic household surveys. Blood donations are sourced countrywide, year-round, and thus provide dynamic, real-time information on the malaria burden. Furthermore, the XN-31 identifies the asymptomatic human reservoir of infectious gametocytes, which must be targeted to eliminate malaria., (© 2022. The Author(s).)

Highly effective vector control can reduce malaria burden significantly, but individuals with parasitemia provide a potential reservoir for onward transmission. We performed an empirical, non-parametric simulation based on cohort data from Tororo District, Uganda-an area with historically high but recently reduced malaria transmission-to estimate the effects of mass drug administration (MDA) and test-and-treat on parasite prevalence. We estimate that a single round of MDA would have accelerated declines in parasite prevalence dramatically over 2 years (cumulative parasite prevalence ratio [PPR], 0.34). This decline was mostly during the first year of administration (PPR, 0.23) and waned by 23 months (PPR, 0.74). Test-and-treat using a highly sensitive diagnostic had nearly the same effect as MDA at 1 year (PPR, 0.27) and required many fewer treatments. The impact of test-and-treat using a standard diagnostic was modest (PPR, 0.58 at 1 year). Our analysis suggests that in areas experiencing a dramatic reduction in malaria prevalence, MDA or test-and-treat with a highly sensitive diagnostic may be an effective way of reducing or eliminating the infectious reservoir temporarily. However, for sustained benefits, repeated rounds of the intervention or additional interventions are required.

Molecular diagnostic approaches are increasingly included in the diagnostic workup and even in the primary diagnosis of malaria in non-endemic settings, where it is difficult to maintain skillful microscopic malaria detection due to the rarity of the disease. Pathogen-specific nucleic acid amplification, however, bears the risk of overlooking other pathogens associated with febrile illness in returnees from the tropics. Here, we assessed the discriminatory potential of metagenomic sequencing for the identification of different Plasmodium species with various parasitemia in EDTA blood of malaria patients. Overall, the proportion of Plasmodium spp.-specific sequence reads in the assessed samples showed a robust positive correlation with parasitemia (Spearman r = 0.7307, p = 0.0001) and a robust negative correlation with cycle threshold (Ct) values of genus-specific real-time PCR (Spearman r = -0.8626, p ≤ 0.0001). Depending on the applied bioinformatic algorithm, discrimination on species level was successful in 50% (11/22) to 63.6% (14/22) instances. Limiting factors for the discrimination on species level were very low parasitemia, species-depending lacking availability of reliable reference genomes, and mixed infections with high variance of the proportion of the infecting species. In summary, metagenomic sequencing as performed in this study is suitable for the detection of malaria in human blood samples, but the diagnostic detection limit for a reliable discrimination on species level remains higher than for competing diagnostic approaches like microscopy and PCR.

Purpose: Babesiosis is one of the most important globally extended and quickly spreading tick-borne infections of dogs. Diagnosis of babesiosis in Sri Lanka is based on clinical signs followed by thin blood smears which could be error-prone due to undetected early infections, absence of clinical signs or low parasitemia. The present study investigated the prevalence of babesiosis in dogs presented to the Veterinary Teaching Hospital (VTH) at the Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, for treatments, vaccinations, and regular check-ups, and compared the diagnosis methods of microscopy and molecular analysis., Methods: Blood samples from dogs were collected from January to June 2019. First, Giemsa stained blood smears were prepared, and then the blood samples were subjected to PCR using genus-specific primers to amplify a 411-450 bp region in the 18S rRNA gene. Twenty samples from PCR amplified products were sequenced for species identification and phylogenetic analysis. Clinical signs of the dogs were noted down, and ticks were also collected from dogs if any., Results: Results show a very high prevalence of canine babesiosis (78.6%) among the dogs brought to the VTH. The parasite was identified microscopically and genetically as Babesia gibsoni. A large percentage (66.7%) of infections was asymptomatic. Out of 42 blood samples, 19 (45.2%) were microscopically positive for babesiosis while 33 (78.6%) were PCR positive, showing a significant difference in the two methods of diagnosis (chi-square test, χ 2  = 9.462, p = 0.002). Three tick species: Rhipicephalus haemaphysaloides, Haemaphysalis bispinosa, and Rhipicephalus sanguineus were found attached to the dogs., Conclusion: This study shows a very high prevalence of canine babesiosis among dogs in the Kandy area. Most of these infections might go undetected if only microscopy was used to diagnose. An improved, rapid diagnostic method such as the novel, PCR-based point-of-care diagnostic method that detects very low parasitemia within 30 min is needed. Moreover, as most infected dogs did not show clinical signs, they may act as reservoirs of infection. The ability of asymptomatic dogs to spread babesiosis should be investigated., (© 2022. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

LAMP diagnosis of malaria is simple and cost-effective with acceptable sensitivity and specificity as compared to standard diagnostic modules such as microscopy, RDTs and nested PCR, and thus its deployment for onsite screening of malaria in resource-limited regions is under consideration. However, the requirement of an electricity-operated dry bath and bulky read-out unit is still a major concern. In an effort to simplify this limitation, we have developed a portable LAMP device and fluorescence readout unit which can be used in the rapid point-of-care diagnosis of malaria. We have developed a point-of-care diagnostic LAMP device that is easy to operate by a mobile application, and the results can be quantified with a fluorescent readout unit. The diagnostic performance of the device was evaluated in 90 P . falciparum -infected clinical isolates stored at 4°C for 6-7 years and 10 freshly collected isolates from healthy volunteers. The LOD and quantitative ability of LAMP in estimating parasitemia levels were revealed with laboratory-grown P. falciparum strain (3D7). The LAMP assay performed in our device was exclusive for P. falciparum detection with sensitivity and specificity determined to be 98.89% and 100%, respectively, in clinical isolates. The LOD was documented to be 1 parasite/µl at the cut-off ADC value of 20. Parasite density estimated from ADC values showed concordance with microscopically determined parasite density of the cultured P. falciparum 3D7 strain. The LAMP assay performed in our device provides a possible portable platform for its deployment in the point-of-care diagnosis of malaria. Further validation of the quantitative ability of the assay with freshly collected or properly stored clinical samples of known parasitemia is necessary for field applicability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Puri, Kaur Brar, Madan, Srinivasan, Rawat, Gorthi, Kumari, Sah, Ojha, Panigrahi, Dhangadamajhi, Muthuswami, Singh and Madhubala.)

Background: Malaria is an infectious disease considered as one of the biggest causes of mortality in endemic areas. This life-threatening disease needs to be quickly diagnosed and treated. The standard diagnostic tools recommended by the World Health Organization are thick blood smears microscopy and immuno-chromatographic rapid diagnostic tests. However, these methods lack sensitivity especially in cases of low parasitaemia and non-falciparum infections. Therefore, the need for more accurate and reliable diagnostic tools, such as real-time polymerase chain reaction based methods which have proven greater sensitivity particularly in the screening of malaria, is prominent. This study was conducted at the French National Malaria Reference Centre to assess sensitivity and specificity of two commercial malaria qPCR kits and two in-house developed qPCRs compared to LAMP., Methods: 183 blood samples received for expertise at the FNMRC were included in this study and were subjected to four different qPCR methods: the Biosynex Ampliquick ® Malaria test, the BioEvolution Plasmodium Typage test, the in-house HRM and the in-house TaqMan qPCRs. The specificity and sensitivity of each method and their confidence intervals were determined with the LAMP-based assay Alethia® Malaria as the reference for malaria diagnosis. The accuracy of species diagnosis of the Ampliquick ® Malaria test and the two in-house qPCRs was also evaluated using the BioEvolution Plasmodium Typage test as the reference method for species identification., Results: The main results showed that when compared to LAMP, a test with excellent diagnostic performances, the two in-house developed qPCRs were the most sensitive (sensitivity at 100% for the in-house TaqMan qPCR and 98.1% for the in-house HRM qPCR), followed by the two commercial kits: the Biosynex Ampliquick ® Malaria test (sensitivity at 97.2%) and the BioEvolution Plasmodium Typage (sensitivity at 95.4%). Additionally, with the in-house qPCRs we were able to confirm a Plasmodium falciparum infection in microscopically negative samples that were not detected by commercial qPCR kits. This demonstrates that the var genes of P. falciparum used in these in-house qPCRs are more reliable targets than the 18S sRNA commonly used in most of the developed qPCR methods for malaria diagnosis., Conclusion: Overall, these results accentuate the role molecular methods could play in the screening of malaria. This may represent a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine analysis, which could be essential for the detection and treatment of malaria carriers and even for the eradication of this disease., (© 2022. The Author(s).)

Background: In Papua (Indonesia), infants with P. falciparum and/or P. vivax malaria are at risk of severe anaemia and death. We hypothesized that in an area of high malaria transmission, intermittent screening and treatment of infants with malaria (ISTi) will reduce morbidity compared to passive case detection (PCDi)., Methods: We conducted a cluster randomised, open label, superiority trial. A total of 21 clusters of village health posts (VHP) were randomised 1:1 to either IST for infants coinciding with 4 routine immunisation visits or PCDi. Healthy term infants born to consenting mothers enrolled into a maternal malaria cluster randomised trial were included in the study and followed for 12 months. Point of care malaria rapid diagnostic tests were used to detect peripheral parasitaemia at 2, 3, 4 and 9 months old in all infants in ISTi clusters and when symptomatic in PCDi clusters. Infants with detected peripheral parasitaemia were treated with dihydroartemisinin-piperaquine. The co-primary outcomes were the incidence rate of clinical malaria in the first year of life and the prevalence of parasitaemia at age 12 months. The incidence rate ratio and prevalence ratio between ISTi and PCDi were estimated using mixed-effects Poisson and log-binomial regression modelling (accounting for clustering at VHP level)., Results: Between May 2014 and February 2017, 757 infants were enrolled into the study, 313 into 10 ISTi clusters, and 444 into 11 PCDi clusters. Overall, 132 episodes of parasitaemia were detected, of whom 17 (12.9%) were in symptomatic infants. Over 12 months, the incidence rate (IR) of clinical malaria was 24 [95% CI, 10-50] per 1000 children-years at risk in the ISTi arm and 19 [95% CI, 8,38] per 1000 children-years in the PCDi arm (adjusted incidence rate ratio [aIRR] 1.77 [95% CI, 0.62-5.01]; p = 0.280). The prevalence of parasitaemia at 12 months was 13% (33/254) in the IST clusters and 15% (57/379) in the PCD clusters (adjusted prevalence ratio (aPR) = 0.92 (95% CI, 0.70-1.21), p = 0.55). There was no difference in the risk of anaemia between treatment arms., Conclusions: In high malaria transmission area outside of Africa, our study suggests that compared to PCDi, ISTi offers no significant benefit in reducing the risk of clinical malaria in infants born to women receiving effective protection from malaria during pregnancy., Trial Registration: ClinicalTrials.gov NCT02001428 , registered on 20 Nov 2013., (© 2022. The Author(s).)

Background: Microscopic examination of Giemsa-stained blood films remains the reference standard for malaria parasite detection and quantification, but is undermined by difficulties in ensuring high-quality manual reading and inter-reader reliability. Automated parasite detection and quantification may address this issue., Methods: A multi-centre, observational study was conducted during 2018 and 2019 at 11 sites to assess the performance of the EasyScan Go, a microscopy device employing machine-learning-based image analysis. Sensitivity, specificity, accuracy of species detection and parasite density estimation were assessed with expert microscopy as the reference. Intra- and inter-device reliability of the device was also evaluated by comparing results from repeat reads on the same and two different devices. This study has been reported in accordance with the Standards for Reporting Diagnostic accuracy studies (STARD) checklist., Results: In total, 2250 Giemsa-stained blood films were prepared and read independently by expert microscopists and the EasyScan Go device. The diagnostic sensitivity of EasyScan Go was 91.1% (95% CI 88.9-92.7), and specificity 75.6% (95% CI 73.1-78.0). With good quality slides sensitivity was similar (89.1%, 95%CI 86.2-91.5), but specificity increased to 85.1% (95%CI 82.6-87.4). Sensitivity increased with parasitaemia rising from 57% at < 200 parasite/µL, to ≥ 90% at > 200-200,000 parasite/µL. Species were identified accurately in 93% of Plasmodium falciparum samples (kappa = 0.76, 95% CI 0.69-0.83), and in 92% of Plasmodium vivax samples (kappa = 0.73, 95% CI 0.66-0.80). Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% of slides., Conclusions: The performance of the EasyScan Go in parasite detection and species identification accuracy fulfil WHO-TDR Research Malaria Microscopy competence level 2 criteria. In terms of parasite quantification and false positive rate, it meets the level 4 WHO-TDR Research Malaria Microscopy criteria. All performance parameters were significantly affected by slide quality. Further software improvement is required to improve sensitivity at low parasitaemia and parasite density estimations. Trial registration ClinicalTrials.gov number NCT03512678., (© 2022. The Author(s).)

Background: Due to increased travel from endemic countries, malaria occurs more frequently in non-endemic regions. It is a challenge for diagnostic laboratories in non-endemic countries to provide reliable results, as experience of staff is often limited to only a few cases per year. This study evaluated the diagnostic accuracy of the fully automated Sysmex XN-31 malaria analyzer in a routine diagnostic setting in a non-endemic region was evaluated., Methods: Samples from 112 patients suspected for malaria were examined by the Sysmex XN-31 analyzer to determine the absolute count of malaria-infected red blood cells count (MI-RBC/µL). Microscopic examination of both Quantitative Buffy Coat capillary tubes and thick and thin blood films were used as reference methods. Limits of blank (LoB), detection (LoD) and quantification (LoQ) were investigated using an in vitro Plasmodium falciparum culture. Nine hundred twenty samples of patients with RBC abnormalities were included to determine which RBC abnormalities trigger indeterminate or false positive results., Results: No false positive nor false negative results were obtained for the examined patient samples suspected for malaria. For 3% of samples an indeterminate result by the XN-31 was obtained. The Passing-Bablok regression line for diagnostic accuracy of the parasitaemia was y = 39.75 + 0.7892 × showing a positive bias of about 21% when comparing the MI-RBC results to microscopy. The LoB, LoD and LoQ were calculated to be 4.7, 5.9, and 19.0 infected RBC/μL, respectively. From the 920 abnormal RBC samples collected, 4.6% resulted in a false positive MI-RBC result and almost half of the samples produced indeterminate results. These results were related to increases in nucleated red blood cells, reticulocytes and other abnormal RBC morphologies such as sickle cells., Conclusions: Based on the results, the XN-31 is a fast and reliable screening method in the detection and quantification of Plasmodium species in patients However, if an abnormal red blood cell morphology is present, the results of the XN-31 should be interpreted with caution as false positive results can be caused by interfering abnormal erythrocytes., (© 2022. The Author(s).)

Background: Some studies have suggested the importance of vitamin D [25(OH)D] in malaria clinical practice. The prevalence of 25(OH)D deficiency in the Amazon population is not well known, and there are few studies in patients with malaria. This study aimed to evaluate 25(OH)D serum levels in patients with malaria and determine their relationships with epidemiological, clinical, laboratory, and parasitemia data., Methods: An analytical cross-sectional study of 123 patients with malaria and 122 individuals without malaria was performed in Itaituba, Pará, Brazil, from January 2018 to October 2019, by evaluating sociodemographic, clinical-epidemiological, parasitological, and laboratory data and adopting a 5% significance level. Parametric tests (Student's t-test), non-parametric tests (Mann-Whitney U), and Spearman's correlation ([rs], for non-parametric variables) were used according to the nature of the distribution of the variables. For the qualitative variables, Pearson's chi-square test, Fisher's exact test, and the G test were used. Spearman's correlation was used to compare the results of the 25(OH)D levels and blood counts performed among patients and the control group., Results: Malaria patients residing in a mining area had 25(OH)D serum levels that were significantly lower than those in the control group residing in the mining area, though both were within normal levels. Red blood cell counts had an inverse correlation with parasitemia (Plasmodium falciparum), and platelet levels had an inverse correlation with parasitemia (Plasmodium vivax). 25(OH)D deficiency was evidenced in Itaituba, in the state of Pará, which is an endemic area of malaria in the Amazon region.

Malaria is often most endemic in remote regions where diagnostic microscopy services are unavailable. In such regions, the use of rapid diagnostic tests fails to quantify parasitemia measurements which reflect the concentration of Plasmodium parasites in the bloodstream. Thus, novel diagnostic and monitoring technologies capable of providing such information could improve the quality of treatment, monitoring, and eradication efforts. A low-cost, portable microscope for gathering quantitative parasitemia data from fluorescently stained thin blood smears is presented. The system employs bimodal imaging using components optimized for cost savings, system robustness, and optical performance. The microscope is novel for its use of monochromatic visible illumination paired with a long working distance singlet aspheric objective lens that can image both traditionally mounted and cartridge-based blood smears. Eight dilutions of red blood cells containing laboratory cultured wild-type P. falciparum were used to create thin smears which were stained with SYBR Green-1 fluorescent dye. Two subsequent images are captured for each field-of-view, with brightfield images providing cell counts and fluorescence images providing parasite localization data. Results indicate the successful resolution of sub-micron sized parasites, and parasitemia measurements from the prototype microscope display linear correlation with measurements from a benchtop microscope with a limit of detection of 0.18 parasites per 100 red blood cells., Competing Interests: The authors have declared that no competing interests exist.

Murine infection models are needed to develop therapeutics and vaccines to combat the Plasmodium parasites causing malaria. Herein, we describe an easy to perform flow cytometry-based methodology for detecting green fluorescent protein-expressing Plasmodium berghei in the peripheral red blood cells (RBC) of mice. This methodology uses one-step staining and simplified gating strategies to streamline the process of Plasmodium quantification and can detect parasitemia at an earlier time point after infection compared to traditional light microscopy-based techniques., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Malaria is a parasitic disease of public health concern especially among children because of their vulnerability. Objective: The study sought to evaluate the prevalence and severity of malaria and to assess the factors associated with malaria parasitaemia among children., Methods: This was a hospital-based cross-sectional observation study. We enrolled 303 children aged 6 to 59 months who presented with fever. A structured questionnaire was used to obtain the demographic characteristics, determinant factors and the use of malaria preventive measures. Microscopic examination of blood film for malaria parasite was done. Data were analysed using SPSS version 23.0. The Pearson's Chi-square was used to determine the association between selected socio-demographic variables, clinical characteristics of participants and presence of malaria parasitaemia. A p-value less than 0.05 was considered significant., Results: The mean ± SD age was 24.36 ± 16.63 months, 183 (60.4%) were male. Two hundred and thirteen (70.3%) participants tested positive for Plasmodium falciparum. Severe malaria accounted for 21.1% of all malaria cases. Severe anaemia (37.8%) and cerebral malaria (24.4%) were the common complications observed. Malaria was significantly associated with increasing age (p = 0.007). Children who slept regularly under LLIN and those using insecticidal spray were more likely to be protected from developing malaria (p = 0.002, p = 0.001, respectively). The socioeconomic status, maternal education, family size and knowledge of LLIN were not associated with the development of malaria (p= 0.901, 0.136, 0.413, 0.166, respectively)., Conclusion: The prevalence of malaria is high; there is a need to increase the coverage of IRS, together with LLINs to reduce the transmission and burden of malaria, particularly among the susceptible population., Competing Interests: The Authors declare that no competing interest exists., (Copyright © 2022 by West African Journal of Medicine.)

Background: Regulatory T cells are known to play a key role to counter balance the protective immune response and immune mediated pathology. However, the role of naturally occurring regulatory cells CD4 + CD25 + Foxp3 + in malaria infection during the disease pathogenesis is controversial. Beside this, ICOS molecule has been shown to be involved in the development and function of regulatory T cell enhance IL-10 production. Therefore, possible involvement of the ICOS dependent regulatory CD4 + ICOS + Foxp3 + T cells in resistance/susceptibility during malaria parasite is explored in this study., Methods: 5 × 10 5 red blood cells infected with non-lethal and lethal parasites were inoculated in female Balb/c mice by intra-peritoneal injection. Infected or uninfected mice were sacrificed at early (3rd day post infection) and later stage (10th day post infection) of infection. Harvested cells were analysed by using flow cytometer and serum cytokine by Bioplex assay., Results: Thin blood films show that percentages of parasitaemia increases with disease progression in infections with the lethal malaria parasite and mice eventually die by day 14th post-infection. Whereas in case of non-lethal malaria parasite, parasitaemia goes down by 7th day post infection and gets cleared within 13th day. The number of CD4 + ICOS + T cells increases in lethal infection with disease progression. Surprisingly, in non-lethal parasite, ICOS expression decreases after day 7th post infection as parasitaemia goes down. The frequency of CD4 + ICOS + FoxP3 + Tregs was significantly higher in lethal parasitic infection as compared to the non-lethal parasite. The level of IL-12 cytokine was remarkably higher in non-lethal infection compared to the lethal infection. In contrast, the level of IL-10 cytokines was higher in lethal parasite infection compared to the non-lethal parasite., Conclusion: Taken together, these data suggest that lethal parasite induce immunosuppressive environment, protecting from host immune responses and help the parasite to survive whereas non-lethal parasite leads to low frequencies of Treg cells seldom impede immune response that allow the parasite to get self-resolved., (© 2022. The Author(s).)

Background: Malaria is endemic in many states of India. Though there are reports of maternal and congenital malaria from endemic areas, however, there remains a paucity of data from hilly terrains. The present study evaluated the prevalence, clinical and microbiological spectrum of maternal and congenital malaria at a tertiary health care facility in Northern India over a period of 18 months., Methods: In this observational study, mothers along with their newborns were evaluated for malaria by maternal, placental, and cord blood smear examination and rapid point-of-care diagnostic serological tests. Positive cases were confirmed by polymerase chain reaction. Mother-newborn duos were followed up till discharge from the hospital., Results: A total of 843 mothers delivered during the study period and were screened along with their newborns and placentae. A total of Ten (1.18%) mothers had evidence of malarial parasitemia (Plasmodium vivax, n=7 and Pl. falciparum, n=3), however, none of the placental and cord blood samples were positive for malaria. Overall, 127 (15.1%) neonates required admission in neonatal intensive care unit for various morbidities. Incidence of small for gestational age (SGA) was high (n=210; 24.9%). Multivariate logistic regression analysis demonstrated maternal malaria to be an independent contributor for SGA [Odds Ratio (95% Confidence Interval), 10.7 (2.06 - 49.72)]. However, only 2% variance of SGA could be explained by maternal malaria alone., Conclusion: We report an encouragingly lower incidence of maternal malaria in mothers attending for delivery and a 'Zero' incidence for placental and congenital malaria during the study period as compared to national data (upto 7.4% in non-immune mothers), although maternal malaria could be a causative factor for SGA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Estimating malaria parasite density is important for patient care and management in rural and urban health centers in endemic areas. Here, we describe methodologically the protocols and methods to identify and enumerate Plasmodium falciparum parasites from infected blood using light microscopy. We provide step-by-step protocols and evaluate any possible drawbacks that may limit the methods and prospects of using light microscopy in diagnosing malaria., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Clinical babesiosis is diagnosed, and parasite burden is determined, by microscopic inspection of a thick or thin Giemsa-stained peripheral blood smear. However, quantitative analysis by manual microscopy is subject to error. As such, methods for the automated measurement of percent parasitemia in digital microscopic images of peripheral blood smears could improve clinical accuracy, relative to the predicate method., Methods: Individual erythrocyte images were manually labeled as "parasite" or "normal" and were used to train a model for binary image classification. The best model was then used to calculate percent parasitemia from a clinical validation dataset, and values were compared to a clinical reference value. Lastly, model interpretability was examined using an integrated gradient to identify pixels most likely to influence classification decisions., Results: The precision and recall of the model during development testing were 0.92 and 1.00, respectively. In clinical validation, the model returned increasing positive signal with increasing mean reference value. However, there were 2 highly erroneous false positive values returned by the model. Further, the model incorrectly assessed 3 cases well above the clinical threshold of 10%. The integrated gradient suggested potential sources of false positives including rouleaux formations, cell boundaries, and precipitate as deterministic factors in negative erythrocyte images., Conclusions: While the model demonstrated highly accurate single cell classification and correctly assessed most slides, several false positives were highly incorrect. This project highlights the need for integrated testing of machine learning-based models, even when models in the development phase perform well., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Microscopic examination of thick and thin blood films is the gold standard in current guidelines for the diagnosis of malaria, but guidelines do not uniformly agree on which combination of other methods should be used and when., Methods: Three questionnaires were sent between March 2018 and September 2019 to laboratories subscribing to the external quality assessment scheme for the diagnosis of blood and intestinal parasites of the Dutch Foundation for Quality Assessment in Medical Laboratories in order to investigate how much variation in the laboratory diagnosis of malaria between different clinical laboratories is present in the Netherlands., Results: The questionnaires were partially or fully completed by 67 of 77 (87%) laboratories. Only 9 laboratories reported 10 or more malaria positive patients per year. Most laboratories use a different diagnostic strategy, within office versus outside office hours depending on the screening assay result. Within office hours, 62.5% (35/56) of the responding laboratories perform an immunochromatographic test (ICT) in combination with microscopic examination of thick and thin blood films without additional examinations, such as Quantitative Buffy Coat and/or rtPCR analysis. Outside office hours 85.7% (48/56) of laboratories use an ICT as single screening assay and positive results are immediately confirmed by thick and thin blood films without additional examinations (89.6%, 43/48). In case of a negative ICT result outside office hours, 70.8% (34/48) of the laboratories perform microscopic examination of the thick film the next morning and 22.9% (11/48) confirm the negative ICT result immediately. Furthermore, substantial differences were found in the microscopic examinations of thick and thin blood films; the staining, theoretical sensitivity of the thick film and determination of parasitaemia., Conclusions: This study demonstrated a remarkably high variation between laboratories in both their diagnostic strategy as well as their methods for microscopic examination for the diagnosis of malaria in a clinical setting, despite existing national and international guidelines. While the impact of these variations on the accuracy of the diagnosis of malaria is yet unknown, these findings should stimulate clinical laboratories to critically review their own diagnostic strategy., (© 2021. The Author(s).)

The scourge of malaria infection continues to strike hardest against pregnant women and children in Africa and South East Asia. For global elimination, testing methods that are ultrasensitive to low-level ring-staged parasitemia are urgently required. In this study, we used a novel approach for diagnosis of malaria infection by combining both electronic ultraviolet-visible (UV/vis) spectroscopy and near infrared (NIR) spectroscopy to detect and quantify low-level (1-0.000001%) ring-staged malaria-infected whole blood under physiological conditions uisng Multiclass classification using logistic regression, which showed that the best results were achieved using the extended wavelength range, providing an accuracy of 100% for most parasitemia classes. Likewise, partial least-squares regression (PLS-R) analysis showed a higher quantification sensitivity ( R 2 = 0.898) for the extended spectral region compared to UV/vis and NIR ( R 2 = 0.806 and 0.556, respectively). For quantifying different-stage blood parasites, the extended wavelength range was able to detect and quantify all the Plasmodium falciparum accurately compared to testing each spectral component separately. These results demonstrate the potential of a combined UV/vis-NIR spectroscopy to accurately diagnose malaria-infected patients without the need for elaborate sample preparation associated with the existing mid-IR approaches.

The co-reactivity of the Plasmodium histidine-rich protein 2 (HRP2) and lactate dehydrogenase (pLDH) in malaria rapid diagnosis tests (mRDTs) as a potential indicator of high parasitemia linked to Plasmodium falciparum was evaluated in the reported study from Cameroon. The samples were screened for malaria using both mRDTs (SD bioline HRP2/pLDH), light microscopy and further confirmed by Plasmodium species-specific PCR assay. Of the 483 patients enrolled, 161 (33.3%) showed a reactive mRDTs amongst which 70 patients were positive by both microscopy and mRDTs with 30.0% (21/70) positive for HRP2 alone, while 70.0% (49/70) showed a dual reaction to HRP2 and pLDH parasite antigens. P. falciparum parasitemia was found to be significantly high among patients with both reactive antigens, (p < 0.0001) suggesting that mRDTs reactivity is influenced by parasite load which could be used as a diagnostic marker for therapeutic management of patients with high parasitemia in field conditions.

Background: Globally, there are over 200 million cases of malaria annually and over 400,000 deaths. Early and accurate detection of low-density parasitaemia and asymptomatic individuals is key to achieving the World Health Organization (WHO) 2030 sustainable development goals of reducing malaria-related deaths by 90% and eradication in 35 countries. Current rapid diagnostic tests are neither sensitive nor specific enough to detect the low parasite concentrations in the blood of asymptomatic individuals., Methods: Here, an imaging-based sensing technique, particle diffusometry (PD), is combined with loop mediated isothermal amplification (LAMP) on a smartphone-enabled device to detect low levels of parasitaemia often associated with asymptomatic malaria. After amplification, PD quantifies the Brownian motion of fluorescent nanoparticles in the solution during a 30 s video taken on the phone. The resulting diffusion coefficient is used to detect the presence of Plasmodium DNA amplicons. The coefficients of known negative samples are compared to positive samples using a one-way ANOVA post-hoc Dunnett's test for confirmation of amplification., Results: As few as 3 parasite/µL of blood was detectable in 45 min without DNA extraction. Plasmodium falciparum parasites were detected from asymptomatic individuals' whole blood samples with 89% sensitivity and 100% specificity when compared to quantitative polymerase chain reaction (qPCR)., Conclusions: PD-LAMP is of value for the detection of low density parasitaemia especially in areas where trained personnel may be scarce. The demonstration of this smartphone biosensor paired with the sensitivity of LAMP provides a proof of concept to achieve widespread asymptomatic malaria testing at the point of care., (© 2021. The Author(s).)

This cross-sectional study evaluated epidemiologic characteristics of persons living with HIV (PWH) coinfected with Trypanosoma cruzi in Cochabamba, Bolivia, and estimated T. cruzi parasitemia by real-time quantitative polymerase chain reaction (qPCR) in patients with and without evidence of reactivation by direct microscopy. Thirty-two of the 116 HIV patients evaluated had positive serology for T. cruzi indicative of chronic Chagas disease (27.6%). Sixteen of the 32 (50%) patients with positive serology were positive by quantitative polymerase chain reaction (qPCR), and four of the 32 (12.5%) were positive by direct microscopy. The median parasite load by qPCR in those with CD4+ < 200 was 168 parasites/mL (73-9951) compared with 28.5 parasites/mL (15-1,528) in those with CD4+ ≥ 200 (P = 0.89). There was a significant inverse relationship between the degree of parasitemia estimated by qPCR from blood clot and CD4+ count on the logarithmic scale (rsBC= -0.70, P = 0.007). The correlation between T. cruzi estimated by qPCR+ blood clot and HIV viral load was statistically significant with rsBC = 0.61, P = 0.047. Given the significant mortality of PWH and Chagas reactivation and that 57% of our patients with CD4+ counts < 200 cells/mm3 showed evidence of reactivation, we propose that screening for chronic Chagas disease be considered in PWH in regions endemic for Chagas disease and in the immigrant populations in nonendemic regions. Additionally, our study showed that PWH with advancing immunosuppression have higher levels of estimated parasitemia measured by qPCR and suggests a role for active surveillance for Chagas reactivation with consideration of treatment with antitrypanosomal therapy until immune reconstitution can be achieved.

Manual microscopic inspection of fixed and stained blood smears has remained the gold standard for Plasmodium parasitemia analysis for over a century. Unfortunately, smear preparation consumes time and reagents, while manual microscopy is skill-dependent and labor-intensive. Here, we demonstrate that deep learning enables both life stage classification and accurate parasitemia quantification of ordinary brightfield microscopy images of live, unstained red blood cells. We tested our method using both a standard light microscope equipped with visible and near-ultraviolet (UV) illumination, and a custom-built microscope employing deep-UV illumination. While using deep-UV light achieved an overall four-category classification of Plasmodium falciparum blood stages of greater than 99% and a recall of 89.8% for ring-stage parasites, imaging with near-UV light on a standard microscope resulted in 96.8% overall accuracy and over 90% recall for ring-stage parasites. Both imaging systems were tested extrinsically by parasitemia titration, revealing superior performance over manually-scored Giemsa-stained smears, and a limit of detection below 0.1%. Our results establish that label-free parasitemia analysis of live cells is possible in a biomedical laboratory setting without the need for complex optical instrumentation. We anticipate future extensions of this work could enable label-free clinical diagnostic measurements, one day eliminating the need for conventional blood smear analysis., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The authors declare provisional patent application 63/072,037 filed on 08/28/2020.

Biomarkers (coming from host or parasite) to monitor Chagas disease (CD) progression as well as the therapeutic response in chronic CD are critically needed, since seronegativization, which may be considered the best indicator of therapeutic cure, takes several years to be observed in adults. Several molecules have been suggested as biomarkers for CD, however, they have to be validated. Taking advantage of mouse models of Trypanosoma cruzi infection, we investigated changes in the degradation profile of fibronectin in plasma. The degradation profile of fibronectin was different in the acute phase compared to the chronic phase of the infection. Fibronectin fragments of approximately 150, 100, 40 and 30 kDa were identified. Furthermore, those degradation profiles correlated with acute parasitaemia as well as with cardiac parasite burden and tissue damage during the infection. The usefulness of fibronectin degradation as a biomarker for therapeutic response following drug treatment and immunotherapeutic vaccination also was evaluated and a decreased fibronectin degradation profile was observed upon benznidazole or a vaccine candidate treatment.

New point-of-care diagnostic approaches for malaria that are sensitive to low parasitemia, easy to use in a field setting, and affordable are urgently required to meet the World Health Organization's objective of reducing malaria cases and related life losses by 90% globally on or before 2030. In this study, an inexpensive "matchbox size" near-infrared (NIR) spectrophotometer was used for the first time to detect and quantify malaria infection in vitro from isolated dried red blood cells using a fingerpick volume of blood. This the first study to apply a miniaturized NIR device to diagnose a parasitic infection and identify marker bands indicative of malaria infection in the NIR region. An NIR device has many advantages including wavelength accuracy and repeatability, speed, resolution, and a greatly improved signal-to-noise ratio compared to existing spectroscopic options. Using multivariate data analysis, we discriminated control red blood cells from infected cells and established the limit of detection of the technique. Principal component analysis displayed a good separation between the infected and uninfected RBCs, while partial least-squares regression analysis yielded a robust parasitemia prediction with root-mean-square error of prediction values of 0.446 and 0.001% for the higher and lower parasitemia models, respectively. The R 2 values of the higher and lower parasitemia models were 0.947 and 0.931, respectively. Finally, an estimated parasitemia detection limit of 0.00001% and a qunatification limit of 0.001% was achieved; to ascertain the true efficacy of the technique for point-of-care screening, clinical studies using large patient numbers are required, which is the subject of future studies.

In November 2018, we diagnosed a cluster of falciparum malaria cases in three Chilean travelers returning from Nigeria. Two patients were treated with sequential intravenous artesunate plus oral atovaquone/proguanil (AP) and one with oral AP. The third patient, a 23-year-old man, presented with fever on day 29 after oral AP treatment and was diagnosed with recrudescent falciparum malaria. The patient was then treated with oral mefloquine, followed by clinical recovery and resolution of parasitemia. Analysis of day 0 and follow-up blood samples, collected on days 9, 29, 34, 64, and 83, revealed that parasitemia had initially decreased but then increased on day 29. Sequencing confirmed Tyr268Cys mutation in the cytochrome b gene, associated with atovaquone resistance, in isolates collected on days 29 and 34 and P. falciparum dihydrofolate reductase mutation Asn51Ile, associated with proguanil resistance in all successfully sequenced samples. Molecular characterization of imported malaria contributes to clinical management in non-endemic countries, helps ascertain the appropriateness of antimalarial treatment policies, and contributes to the reporting of drug resistance patterns from endemic regions.

Objective: In sub-Saharan Africa where 90% of malaria cases are concentrated, the control of this disease constitutes a major challenge whose diagnosis by thick and thin smear deserves to be exact and reproducible. The purpose of this study is to assess the performance of thick/thin blood smear in order to improve its implementation process., Material and Methods: This was a descriptive and analytical study that took place from May to June 2017 and involved participating laboratories (PL) coming from public, liberal and confessional sectors in Lomé. A set of 13 blood smear slides of variable parasite densities (PD) with assigned values (AV) of parasite densities and the Plasmodium species assigned was used. The criterion for establishing the parasite densities compliance interval was assigned values ± 25% and the performance rates were compared to the 80% recommended by the WHO for Africa region., Results: 41.9% (13/31) of the PLs had a compliance rate greater than 80% including four with a performance of 100% for the ability to identify the Plasmodium species. For the parasitaemia < 100/μl, 51.6% of participating laboratories had a performance rate less than 80% and for parasitaemia > 2000/μl, 100% of these laboratories had a performance rate greater than 80%., Conclusion: The evaluated laboratories had insufficient ability for the identification of Plasmodium falciparum and the correct estimation of low parasitaemia. A need to strength the technical skills, adapted to the context of low parasitaemia are essential to improve the biological diagnosis of malaria in Togo., Competing Interests: Les auteurs ne déclarent aucun conflit d'intérêts., (Copyright © 2021 SFMTSI.)

The automated hematology analyzers XN-30 (for research) and XN-31 prototype (for diagnosis support) can easily and rapidly detect Plasmodium-infected red blood cells (iRBCs) and distinguish the developmental stages of the parasite in approximately 1 min. Two dedicated reagents, Lysercell M and Fluorocell M, are available with the analyzers. Lysercell M plays an indispensable role in enhancing the fluorescence intensity of the nucleic acid staining dye in Fluorocell M and altering cell morphology. These effects of Lysercell M have been empirically determined but insufficiently analyzed. In this study, the properties of Lysercell M were analyzed using two flow cytometers and a fluorescence microscope. First, the fluorescence intensity emitted by iRBCs treated with Lysercell M or phosphate-buffered saline (PBS) was evaluated. Second, the size of RBCs treated with Lysercell M or PBS was measured. Finally, the morphology of individual parasites was observed after reconstruction of an M scattergram, a cytogram of the XN-31 prototype system, using an imaging flow cytometer. These analyses showed that treatment of iRBCs with Lysercell M increased the fluorescence intensity of stained parasite nucleic acids by approximately 10-fold and reduced the size of iRBCs in a stage-specific manner, facilitating the identification and quantification of ring form, trophozoite, and schizont stage iRBCs. These properties suggest that Lysercell M is useful for rapidly detecting iRBCs and accurately distinguishing the parasite developmental stages, thereby contributing to the usability of the XN-30 and XN-31 prototype analyzers., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Despite many technological advances for malaria parasite detection (e.g. high resolution image acquisition), microscopic reading of thick blood smear (TBS) remains the gold standard. Even though available in low technology environment, the microscopy of TBS is slow and time consuming. Moreover microscopy may induce errors at many levels and has no quality control., Methods: A electronic extension of the mechanical tally counter is proposed. In addition to the counting process it includes the process of counting itself that relies on the time elapsed between two successive pressures of the counting button leading to a timed tally counter (TTC). The microscopist performs the reading with the specific instruction starting by counting, in each high power fields, leucocytes first and then parasites. The time-stamp of all pressures of counting buttons are recorded along with the nature of the count. The data are recorded internally in CSV format and are exportable. The detection of HPFs locations and leukocyte/parasite counts per HPFs is performed through a hidden semi-Markov model (with outliers) allowing both to take into account the known distribution of leukocyte per HPFs (using a negative binomial distribution) and the pauses and hesitation of the microscopist during the reading. Parameters are estimated via the expectation-maximization algorithm. Hyper-parameters are calibrated using expert annotations. Forward/backward recursions are used to obtain the HPFs locations., Results: This approach provides richer data at no extra cost. It has been demonstrated that the method can derive parasites per HPF, leukocytes per HPF, and parasite/leukocyte ratio with robust non-parametric confidence intervals. Moreover a direct digital data entry leads to a less expensive process and decreased time-consuming and error-prone manual data entry. Lastly the TTC allows detecting possible protocol break during reading and prevents the risk of fraud., Discussion and Conclusion: Introducing a programmed digital device in the data acquisition of TBS reading gives the opportunity to develop easily new (possible adaptive) reading protocols that will be easily followed by the reader since they will be embedded directly in the device. With the TTC the reader only has to read HPFs, counting leukocytes first and parasites second, and the counter will beep when the protocol is completed.